The current invention is directed toward a method for identifying components involved in signal transduction pathways in higher plants. In particular, the present invention relates to a method to elucidate key nucleic acid sequences of components involved in the signal transduction pathway that communicates mitochondrial function and metabolic status to nuclear gene expression. The present invention also relates to a method for identifying key nucleic acid sequences of components in the signal transduction pathway between branched chain amino acid biosynthetic pathways and nuclear gene expression. Additionally, the present invention also relates to a polynucleotide that encodes a portion of an AOX promoter, AOX1a, operably linked to a luciferase reporter gene. The present invention also relates to a recombinant vector, transformed cells, and transformed organisms containing this polynucleotide.
Mitochondria play a crucial role in the overall physiology of an organism and are a primary site of energy production in all eukaryotic cells. The products of carbohydrate, lipid, and protein catabolism enter the mitochondria and are oxidized by the tricarboxylic acid cycle (TCA) leading to the production of reducing equivalents (NADH and succinate), ATP, and CO2 (Mackenzie et al., (1999) Plant Cell 11:571-586). Energy production occurs as the reducing equivalents NADH and succinate are oxidized and the electrons are fed into a series of enzyme complexes called the mitochondrial electron transport (or respiratory) chain (mtETC). In most eukaryotes, the electrons are passed through the mtETC via the cytochrome respiratory pathway (FIG. 1). In this pathway, electrons move through the mtETC from complex I (NADH dehydrogenase), or complex II (succinate dehydrogenase) or the internal rotenone-insensitive, external and outer membrane NAD(P)H dehydrogenases to the ubiquinone pool. Electrons are then transferred to complex III, then to diffusible cytochrome c and finally to complex IV, which utilizes the electrons to reduce oxygen to water. At several points in the pathway, protons are pumped from the matrix to the intermembrane space, establishing a proton gradient across the inner membrane. The proton gradient is then utilized by complex V (ATPase) to drive the synthesis of ATP. Hence, the cytochrome pathway couples oxidation to the synthesis of ATP. ATP is primary source of chemical energy in the cell.
In higher plants, some fungi, unicellular green algae, and trypanosomes, however, an alternative mtETC pathway exists (referred to as the alternative respiratory pathway) (FIG. 1). In the alternative respiratory pathway, electrons can move from the ubiquione pool to alternative oxidase (AOX), which also reduces oxygen to water (Mackenzie et al., (1999) Plant Cell 11:571-586). AOX does not pump protons and therefore, this pathway results in either a much lower or no establishment of a proton gradient. The end result of the alternative oxidase pathway is an uncoupling of electron transport from ATP synthesis wherein the energy from electron transport is dissipated as heat instead of being harnessed for the production of ATP. The function of the alternative pathway has yet to be fully elucidated, however, proposed functions include 1) an overflow for electrons when the cytochrome pathway is saturated; 2) a means of allowing continued carbon skeleton turnover and conversion when cellular energy is high; and 3) an elimination system for reactive oxygen species. In addition, AOX activity has been shown to be influenced by environmental, developmental, chemical and tissue specific signals (Aubert et al., (1997) Plant J. 11:649-657; and Mackenzie et al., (1999) Plant Cell 11:571-586).
In order to preserve mitochondrial integrity, plants must perceive and respond to numerous developmental changes and environmental stresses. It is of particular importance for plants, that must survive in place, to be able to adapt to harsh environmental conditions. At the molecular level, one mechanism plants and other organisms have in their repertoire to cope with such conditions is the alteration of protein expression. For example, plants alter the expression of proteins in the mtETC as a means to ensure that electron flow is correctly partitioned between the cytochrome and alternative oxidase pathways to meet the energy demands of the cell at any given time. Additionally, numerous environmental stresses and developmental signals can alter the protein profile of mitochondria (Sachs et al., (1986) Ann. Rev. of Plant Physiol. 37:363-376). For example, in response to heat-stress a new class of proteins is induced (the so called xe2x80x9cheat shockxe2x80x9d proteins) to help ameliorate the impact of heat-stress on the plant (Waters et al., (1996) J. Exp. Bot. 47:325-338). Plants also alter protein expression in response to a number of other environmental stresses including but not limited to: phosphate deficiency, cold stress, aging, salt stress and elevated CO2 levels.
The means by which plant mitochondria alter protein expression is complicated and remains largely enigmatic. The mitochondria has a genome of its own, however, only 10% of the genes needed by the mitochondria are encoded by its genome (Schuster et al., (1994) Ann. Rev. Plant Mol. Biol. 45:61-78). Thus, most mitochondrial proteins are the products of nuclear genes that are imported into the mitochondria from the cytosol following their synthesis. The means by which mitochondrial status is communicated to the nucleus is through a signal transduction pathway (de Winde et al., (1993) Saccharomyces cerevisiae. Prog. Nucleic Acid Res. Mol. Biol. 46:51-91; Poyton et al., (1 996) Annu. Rev. Biochem. 65:563-607; and Zitomer et al., (1992) Saccharomyces cerevisiae. Microbiol. Rev. 56:1-11). Signal transduction pathways are one mechanism that the cell uses to respond to the surrounding environment (Lewin B. (1997) Genes VI, Oxford University Press, 1053-1082). Through a series of reactions involving numerous protein components and secondary messengers, signal transduction pathways communicate environmental status to the nucleus so that gene expression is tailored to meet the protein demands resulting from the environmental or developmental changes.
Organisms must also regulate the expression of genes encoding proteins involved in branched chain amino acid biosynthesis and likely accomplish this through signal transduction pathways. Acetolactate synthase catalyzes the first committed step in the pathway and its inhibition leads to metabolic perturbation, which results in a lack of branched chain amino acids (Aubert et al., (1997) Plant J. 11:649-657; and Bryan J. K. (1980) The Biochemistry of Plants: A Comprehensive Treatise, Academic Press 5:403-452). Inhibition of branched chain amino acid biosynthesis has been shown to result in an accumulation of AOX protein and transcript (Aubert et al., (1997) Plant J. 11:649-657). Specifically, the herbicides sulfmometuron methyl, chlorsulfuron and sceptor have all been shown to inhibit branched chain amino acid biosynthesis and result in an increase in AOX transcription (Aubert et al., (1997) Plant J. 11:649-657). Hence, characterizing the signal transduction pathway between branched chain amino acid biosynthetic pathways and nuclear gene expression is of particular interest in plants because of the impact that herbicides can have on overall plant metabolism. For example, understanding the mechanism by which specific metabolic enzyme expression is altered in response to herbicide application may provide a means to control the overall response of plants to herbicides.
Research methods to efficiently and comprehensively determine the mechanism of signal transduction pathways have not been fully developed. Characterizing key components involved in signal transduction pathways is a formidable challenge due to the number of protein components that participate in transmitting the signal, the complex biochemical mechanism by which the signal is transmitted, and the impact of signal on both gene expression and protein translation. And, as such, the components involved in the signal transduction pathway that communicates mitochondrial status to nuclear genes have not been determined. Additionally, the components involved in the signal transduction pathways between branched chain amino acid biosynthetic pathways and nuclear gene expression have also not been characterized.
A number of hybridization-based approaches maybe employed in order to identify components of such signal transduction pathways. The Northern Blot is one such procedure that has been utilized to detect a RNA sequence encoding proteins whose expression is altered. However, this method is extremely laborious and inefficient when the goal of the study is to identify unknown components in a signal transduction pathway leading to altered gene expression. Literally, employing this technique may necessitate probing for every mRNA sequence in each mutant plant to identify the gene with the mutation.
Gene tagging based approaches, such as T-DNA tagging, are other methods that have been employed to determine components of a signal transduction pathway (Walbot V., (1992) Ann. Rev. Plant Phys. Mol. Biol. 43:49-82). The idea behind T-DNA gene tagging is that a mobile or introduced piece of DNA can sometimes insert into a gene , and thereby modify gene expression. These xe2x80x9cmutatedxe2x80x9d genes are now xe2x80x9ctaggedxe2x80x9d with foreign DNA. By using a probe for the introduced DNA tag, one can identify genomic clones that contain the DNA tag, and therefore the gene that is mutated. However, like hybridization based techniques, gene tagging also presents significant shortcomings. The primary shortcoming of this approach for screening mutants in a signal transduction pathway is developing a reliable screening technique. The only phenotype may be altered gene expression and the only way to screen in this case, would again, be to perform Northern Blots for each T-DNA mutant.
In order to overcome these shortcomings, recent approaches to identify components in signal transduction pathways have focused on the use of reporter systems to determine mutants of interest in conjunction with genetic techniques to identify the gene with the mutation. A reporter gene is a coding region, which when expressed displays an easily assayed and novel phenotype or biochemistry in the organism, thus reporting on the activity of a promoter to which it is operably linked. A reporter based approach has been used to identify mutants in the signal transduction pathways in Arabidopsis thaliana for circadian rhythm, osmotic and cold stress (Millar et al., (1995) Science 267:1161-1163and 1163-1166; and Ishitani et al., (1998) Plant Cell 10:1151-1161). However, Millar and Ishitani do not teach or suggest a reporter system to identify components of either the signal transduction pathway that communicates mitochondrial status to nuclear gene expression or the signal transduction pathway between branched chain amino acid biosynthetic pathways and nuclear gene expression.
Among the objects of the present invention is the provision of a method to efficiently and comprehensively identify components involved in these signal transduction pathways that communicate mitochondrial status to nuclear gene expression and between branched chain amino acid pathways and gene expression. The elucidation of key proteins in these signal transduction pathways will allow plants to be genetically engineered for increased productivity, herbicide resistance, pest resistance or increased stress tolerance. The current invention meets this need.
The present invention provides a method to identify components of the signal transduction pathways either between mitochondrial status and nuclear gene expression or between branched chain amino acid biosynthetic pathways and nuclear gene expression. This method, unlike current approaches, provides a means to efficiently and comprehensively identify nucleic acid sequences encoding protein components of the pathways by utilizing a novel reporter system.
Accordingly, among the aspects of the present invention is to provide a method for identifying the nucleic acid sequence of components of the signal transduction pathways between mitochondrial function and metabolic status and nuclear gene expression in higher plants comprising:
(a) transformation of a plant with a vector that encodes a reporter gene operably linked to an AOX promoter;
(b) identification of a transgenic plant that increases the expression of the reporter gene relative to the basal level of endogenous expression of such gene when subjected to a stimuli;
(c) mutagenesis of the transgenic plant identified in step b;
(d) selection of a mutant transgenic plant from step c, wherein such plant exhibits altered expression of the reporter gene; and
(e) determining the identity of a gene from the mutant plant from step d that encodes a protein that participates in such signal transduction pathway.
Another aspect of the invention is a method to identify the nucleic acid sequence of components of the signal transduction pathways between branched chain amino acid biosynthetic pathways and nuclear gene expression in higher plants comprising:
(a) transformation of a plant with a vector that encodes a reporter gene operably linked to an AOX promoter;
(b) identification of a transgenic plant that increases the expression of the reporter gene relative to the basal level of endogenous expression of such gene when subjected to a stimuli;
(c) mutagenesis of the transgenic plant identified in step b;
(d) selection of a mutant transgenic plant from step c, wherein such plant exhibits altered expression of the reporter gene; and
(e) determining the identity of a gene from the mutant plant from step d that encodes a protein that participates in such signal transduction pathway.
Yet another aspect of the invention is a recombinant vector comprising a member selected from the group consisting of:
(a) a polynucleotide which has the nucleic acid sequence comprising bases 361 through 3317 of SEQ ID NO:1. or the complement thereof;
(b) a polynucleotide that has at least 90% sequence identity with the polynucleotide of (a);
(c) a polynucleotide that hybridizes to the polynucleotide of (a) under conditions of 5xc3x97SSC, 50% formamide and 42xc2x0 C., and which encodes a protein having the same biological function;
(d) a polynucleotide encoding the same amino acid sequence as (a), but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code;
(e) a polynucleotide encoding the same amino acid sequence as (b), but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code; and
(f) a polynucleotide encoding the same amino acid sequence as (c), but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code.
Another aspect of the invention is a recombinant polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide comprising bases 361 through 3317 of SEQ ID NO: 1 or the complement thereof;
(b) a polynucleotide that has at least 90% sequence identity with the polynucleotide of (a);
(c) a polynucleotide that hybridizes to the polynucleotide of (a) under conditions of 5xc3x97SSC, 50% from amide and 42xc2x0 C., and which encodes a protein having the same biological function;
(d) a polynucleotide encoding the same amino acid sequence as (a), but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code;
(e) a polynucleotide encoding the same amino acid sequence as (b), but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code; and
(f) a polynucleotide encoding the same amino acid sequence as (c), but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code.
Yet another aspect of the invention is a recombinant host cell transformed with a vector described above.
Another aspect provides an organism transformed with the vector described above.
A further aspect provides a recombinant host cell transformed with a recombinant polynucleotide described above.
In yet another aspect of the invention provides a recombinant organism transformed with a recombinant polynucleotide described above.
Other features of the present invention will be in part apparent to those skilled in the art and in part pointed out in the detailed description provided below.